Meet René Rezsohazy’s lab

The Team, today….

Laure Bridoux, post-doctoral fellow (“Chargée de recherches”, FRS-FNRS ), Damien Marchese, PhD student (Teaching assistant, UCLouvain), Philippe Bombaerts and Raphaël Chiarelli, technicians, members of the AMCB staff.

Ludovic Boas, PhD candidate (“FRIA applicant”, not on the picture), Justine Duphénieux and Georges Delépine, Master students (not on the picture).

Research interests and past achievements

Since their discovery more than 40 years ago, HOX genes have fascinated biologists for their prominent roles in shaping animal body plans in development and evolution[1],[2]. These genes code for transcription factors which have been paradigmatic in understanding the genetic control of development as well as in studying gene regulatory mechanisms.

Approaching developmental processes through the prism of regulations taking place at the protein level has been largely neglected. In fact, the study of HOX proteins and their mode of action has been quite poorly invested. We are among the few research teams worldwide which have started looking at HOX protein activity determinants and interactions[3].

Since the early 2000’s when we established our research group at UCLouvain, Louvain-la-Neuve, we invested this niche consisting in characterizing the molecular biology and interactions of HOX proteins, focusing on two proteins among this family of 39 proteins in mammals, namely HOXA1 and HOXA2.

First, the so-called Three Amino Acid Loop Extension (TALE) homeodomain proteins have been identified in the late 90s as important interactors of HOX proteins contributing to their binding specificity onto DNA. We contributed to reveal that the HOX-PBX interaction is functionally critical. In that regard, we demonstrated in vivo and in vitro that HOXA1 is the HOX family member which most critically relies on this protein partnership[4],[5],[6].

Figure 1. Nuclear interactors of HOXA1 (in orange).

 

Second, we identified molecular determinants of the HOXA1 functional specificity and transcriptional activity[7],[8] and we were the first -and still the only research team worldwide- to characterize a mammalian HOX protein interactome on a proteome-wide scale[9](Figure 1). Thanks to the identification of unexpected interactors, we highlighted that HOXA1 not only acts as a transcription factor but also modulates signal transduction pathways at the level of signaling regulators [10] (Figure 2).

Figure 2. Model for the HOXA1-mediated modulation of the NF-κB pathway in breast cancer (from Taminiau et al., 2016).

Next, we have started to study the functional importance of an intriguing histidine (His) repeat motif in HOXA1 which we identified to be important for several molecular interactions[11],[12],[13]. Dr Stéphane Zaffran (Aix-Marseille Université) who identified variants in this His-repeat to be highly correlated and supposedly causative of cardiac malformations in humans, invited us to collaborate on unravelling the molecular causes of HOXA1 variant dysfunction in heart development. We showed that the His-repeat length is critical both for the stability and activity of HOXA1 in vitro (our group), and in the zebrafish model (S. Zaffran)[14]. Finally, we generated CRISPRed mouse mutant lines displaying 8, 10, 12 or 13 His residues instead of 11 in the His-repeat of the wildtype HOXA1. These lines are currently being characterized for the occurrence and spectrum of cardiac malformations (see below, description of the running projects).

HOXA1 has been identified to be an active oncogene in breast cancers. Our understanding of the molecular biology of HOXA1 allowed us to conclude that its oncogenic activity relies on the integrity of the hexapeptide motif mediating its interaction with PBX as well as on the capacity HOXA1 has to modulate signal transduction so to activate the NFkB transcription factor[15],[16] (Figure 1). This highlighted that the HOXA1 oncogenic activity is multimodal, relying on its ability to interact with PBX and activate transcription, as well as in modulating cell signaling. More recently, we demonstrated that HOXA1 can interact with and antagonize the activity of the estrogen receptor ERα which is a key player in the vast majority of breast cancers[17]. Our work significantly contributed to get an overview about the mode of action of HOXA1 as an oncoprotein[18].

About HOXA2, we identified a few target genes[19] and in particular, we dissected one important functional enhancer which is one component of the cross-regulatory interactions taking place in patterning the hindbrain in the embryo[20]. This enhancer is embedded in an ultra-conserved genomic element and in the coding sequence of the Hoxa2 gene itself, which were unprecedented features for vertebrate enhancers at the time of its discovery.

We also mapped the HOXA2 interactome (which we did not publish as a whole, preferring to report on functionally characterized interactions). We identified that, similarly to HOXA1, HOXA2 has non-transcriptional activities in modulating the stability of interactors like the E3-ubiquitin ligase RCHY1[21],[22]. We unraveled pathways of activity regulation which involve post-translational modifications as well as intra-cellular trafficking of HOXA2[23],[24]. Activity regulation by post-translational modifications defines one of our current projects (see below, description of the running projects)

Our positioning in the “HOX community” while studying new molecular interactors and determinants at the heart of HOX protein activities and functions, allowed us to establish and invest an original niche in the “HOX” research field.

Running projects

Currently, we keep on two main projects. The first one consisting in the PhD thesis project of Damien Marchese, concerns the involvement of HOXA1 in heart development and aims to understand why length variations of the His-repeat motif of the protein lead to cardiac malformations. Damien therefore engaged three lines of investigation. The first resulted in the characterization of molecular properties of the His-repeat variants of HOXA1. The second which is one of the main current objectives of Damien is to understand why is the His-repeat so important to the activity of HOXA1. He identified that this motif is critical for interacting with Cysteine-rich interactors and that these interactions probably rely on the coordination of divalent cations. A third line of research is about the functional and molecular interaction between HOXA1 and actors of the WNT signaling pathway. Damien identified that HOXA1 has an inhibitory influence on WNT signaling targets. At which level of the pathway, through which protein-protein interactions this inhibition takes place is a question we would like to address…Maybe for a new PhD student or post-doc to come?

The second project is the one pursued by Laure Bridoux, postdoc. Basically, Laure is running her own research on the activity modulation of HOXA2. This project originates from the PhD thesis of Laure and that of Noémie Deneyer afterwards. Laure and Noémie identified that some HOXA2 interactors modulate its exit from the nucleus. This appears to be dependent on ubiquitination and phosphorylation events. A key hypothesis is that in the cytoplasm, HOXA2 is de-ubiquitinylated to constitute a store of protein ready to be recruited to enter into the nucleus in response to environmental cues and signalling (Figure 3). Nuclear HOXA2 appears to be ubiquitinylated and ubiquitinylated HOXA2 seems to associate to the chromatin. Would the ubiquitinylated from of HOXA2 be active at the DNA to modulate target gene transcription, this would define a paradigm shift in the field.

Figure 3. Hypothetical model for activity regulation of HOXA2 by its interactors PPP1CB, a phosphatase, and KPC2, a ubiquitin ligase (from Deneyer et al., 2019)

Besides the running projects of Laure and Damien, our research goal to pursue the characterization of the oncogenic activities of HOXA1 is left on the backburner for the moment, but we hope to give this a revival very soon…Maybe for a new PhD student or post-doc to come?